Multiplex analyzer

Plasma levels of IFABP, CCL25, IL‐18, IL‐18 binding protein (IL‐18BP) and LBP were measured in duplicate by enzyme immunoassays (EIA) using commercially available antibodies (R&D Systems, Minneapolis, MN, USA) in a 384 format using a combination of a SELMA (Jena, Germany) pipetting robot and a BioTek (Winooski, VT, USA) dispenser/washer. Absorption was read at 450 nm with wavelength correction set to 540 nm using an EIA plate reader (Bio‐Rad, Hercules, CA, USA). Plasma levels of IL‐1β and IL‐1Receptor antagonist (IL‐1Ra) were analysed using a multiplex cytokine assay (Bio‐Plex Human Cytokine 27‐Plex Panel; Bio‐Rad Laboratories Inc., Hercules, CA, USA). The samples were analysed on a Multiplex Analyzer (Bio‐Rad Laboratories) according to the manufacturer’s instructions.

Blood samples were taken from the saphenous vein after 7 days of treatment, and at the end of the follow-up period, the animals were sacrificed and blood was collected by heart puncture into EDTA-coated or serum tubes (Becton, Dickinson and Company, Puls A/S, Oslo, Norway). The mice were injected subcutaneously with a mixture of Midazolam (Dormicum) and Fentanyl (Hypnorm) for sedation and analgesia before sacrifice was performed with dislocation of the neck. Collected samples were separated by centrifugation at 3000 rpm for 15 min and stored at -70°C awaiting assessment. Plasma human C-peptide, active GLP-1 (i.e. GLP-1 (7–36) amide) and glucagon were measured using a diabetes 4-plex assay (Bio-Plex, Bio-Rad Laboratories, Hercules, CA) analyzed on a Multiplex Analyzer (Bio-Rad Laboratories) following the manufacturer’s description. Circulating human pro-insulin levels were measured in serum samples using an ELISA assay according to the manufacturer’s description (Mercodia AB, Uppsala, Sweden). Serum triglycerides and total cholesterol concentrations were measured using enzymatic colorimetric reagents (Wako, Osaka, Japan) according to the manufacturer’s description.

Plasma levels of tumor necrosis factor (TNFα) and interleukin (IL)-6, IL-8, and IL-12 were analyzed using a multiplex cytokine assay (Bio-Plex Human Cytokine Plex Panel; Bio-Rad Laboratories Inc., Hercules, CA). The samples were analyzed on a Multiplex Analyzer (Bio-Rad Laboratories) according to instructions from the manufacturer.